Traditionally, screening of agents for biological activity is accomplished by placing small amounts of the compound to be tested, either in liquid or solid form, in a number of wells formed in a microtiter plate. As used herein, the term "liquid" refers to pure liquids, as well as liquids containing particulate matter and solvents containing solute. The compound is then exposed to the target of interest, usually a purified protein such as an enzyme or receptor but also possibly a whole cell or non-biologically derived catalyst. The interaction of the test compound with the target is generally measured radiochemically, spectrophotometrically, or fluorometrically. For example, fluorescent probes have been developed which are substrates for enzymes or calcium indicators, pH indicators, amine-reactive or carboxylic acid-reactive, as discussed in the Handbook of Fluorescent Probes and Research Chemicals, 5th ed., R. Haugland and Karen Larison, editor, published by Molecular Probes, Inc., 1994.
Radiochemical measurement is usually considered the most sensitive of the detection methods, followed closely by fluorescence. However, the problems associated with using radioactive material, such as exposure limits, record keeping, and waste management, make this detection method significantly less attractive than detection by florescence. Consequently, the fluorescence measurement technique has gained wide spread acceptance.
In the fluorescence measurement technique, light of a given wave length is directed onto a sample within the well of a microtiter plate. A portion of this light is absorbed by the sample and reemitted at a different, typically longer, wave length, which is then measured. Instrumentation for fluorescence detection is based on conventional 96-well plates. Such instrumentation is available from Dynatech Laboratories, 14340 Sullyfield Circle, Chentilly, Va. 22021, and Packard Instrument Co., 800 Research Park, Meriden Conn. 06450.
The wells of conventional 96-well plates typically have volumes of approximately 400 microliters each. The wells typically have cylindrical walls and either flat, round, or V-shaped bottoms. The plates are conventionally made from a white or black plastic, such as polystrene, polypropelene, or ABS, that has relatively low intrinsic fluorescent properties. While this low level background fluorescence from the plate material is undesirable, it usually presents no major problems in fluorescence detection studies since the fluorescence from the sample in the well is generally orders of magnitude greater than the background fluorescence from the plate. This difference in fluorescence between the plate material and the sample is due both to the large volume of the sample in the well, usually 50-200 microliters, as well as the low surface area to volume ratio of the well in the plate.
The larger the quantity of wells that can be processed in a given batch, the higher the efficiency of the screening process. Consequently, it is desirable to concentrate a large number of wells in each microtiter plate by using microwells, rather than conventional wells. Such concentration of wells also permits very dense storage of collectives of discrete compounds for later testing as films in addressable grid positions, thus reducing the number of plates that must be tested in a complete collection of compounds.
The use of micro volumes in biological screening is also desirable for reasons other than increased throughput. First, reagents, both biologically and chemically derived, are generally expensive and in very limited supply. By decreasing the assay volume, many more test components can be assayed with a given amount of biological target. Second, combinatorial chemistry libraries are made by the sequential addition of small organic building blocks onto an organic scaffold. The scaffold is covalently linked to a solid support structure, such as a Tentagel resin, via an acid, base, or photo-cleavable linker. Such solid supports structures are commonly referred to as "beads" and encompass structures having a variety shapes and sizes. In general, each bead, which is approximately 130 microns in diameter, contains 100 to 200 picomoles of compound. The small amounts of compound found on a single bead requires that the assay of the compound on that bead be performed in small volume. For example, if all 100 picomoles of compound were cleaved from a single bead into the standard 200 microliter assay volume deposited in the 400 microliter well of a 96 well plate, the concentration would be 500 nanomolar, assuming a molecular weight of 500 daltons. This concentration is significantly below the ideal concentration of 10 micromolar that is generally used for screening compounds for biological activity. Also, it is generally desirable to be able to screen the compound at least twice so that the results can be confirmed if the compound tests active in the first assay. In order to reach the 10 micromolar concentration or to screen the compound at least twice, and have enough left over for determination by mass spectroscopy, the compound should be cleaved into less than 5 microliter.
Unfortunately, assay miniaturization creates a number of problems. Reducing the size of the wells increases the difficulty associated with accurately dispensing liquids into them because it becomes increasingly difficult to locate the dispensing device precisely over the center of each well. Inaccurate locating of the dispensing device will result in liquid being dispensed onto the boundary between wells, rather than into the wells themselves. Unfortunately, the wells of conventional microtiter plates are separated by flat, horizontal surfaces upon which liquid can collect if it is not accurately dispensed into the wells. The collection of liquid between wells can create a variety of problems, including partial filling of wells, loss of reagents, and inaccurate mixing and concentration of components. Although collection of liquid between wells can be minimized by the use of dispensing devices capable of highly accurate positioning (e.g., Packard Nanodrop.TM.), such devices are very slow, rendering impractical kinetic assays that require near simultaneous dispensing of agents into each well. Consequently, the difficulty of accurately dispensing liquids into very small wells has limited the ability to incorporate large numbers of wells into a single microtiter plate.
Another problem associated with the use of small volume microwells is that pipetting into each well must be done sufficiently quickly so that evaporation does not significantly change the volume in the well. Conventional liquid handling devices, such as the Tomtech or Sagian, are capable of pipetting volumes as small as 1 microliter and placing the liquid at defined positions. However, it would take several tens of minutes to fill a microwell plate containing 2400 wells to 9600 wells using these devices, by which time the first filled wells would have experienced significant evaporation. Other liquid handling devices based on inkjet printer head technology, available from BioDot and Packard, are capable of pipetting nanoliter volumes but likewise require significant time to pipet directly into a small microwell.
Another problem associated with the use of small microwells arises in connection with fluorescence detection. The use of very small assay volumes results in significantly reduced reemitted light signals, making the technique extremely sensitive to signal detection errors. For example, a microwell having a volume of 0.5 microliters will produce a signal that is only 0.1 to 0.2% of the signal resulting from the use of the well of a conventional 96-well plate. Accurate measurement of fluorescence is also complicated by the intrinsic fluorescence, in at least one region of the spectrum that is useful for detection of biological reactions, of the plastics from which microtiter plates have conventionally been made, as previously discussed. The effect of such background fluorescence is exacerbated in small volume microwells because the well surface area to volume ratio is significantly greater than in conventional 96-well plates. Consequently, while a given level of background fluorescence might be tolerated in a 96-well plate design, it could potentially be larger than the total signal if the well size was reduced to that of a small microwell.
In addition to the problem of background fluorescence associated with the materials from which conventional microtiter plates are made, the geometry of such plates also creates problems in signal detection. In fluorescence measurement techniques, the detection of the reemitted light from the sample within microtiter plate wells is generally done with a charged coupled device (CCD) camera. This technique requires that the surface of the plate be as flat as possible so that the entire surface is in the same focal plane of the camera lens. If a plate were not flat, the wells across the plate would not be in the same focal plane and, consequently, light detection from the wells would not be uniform. This would, in turn, result in errors in determining the relative activity of the assay components in each well. Although conventional microtiter plates feature wells having a variety of bottoms (e.g., flat, V-shaped, round), the well walls of conventionally 96-well microtiter plates are cylindrical. When imaged with a CCD camera, such a cylindrical well act as a lens, which tends to focus the reemitted light from the sample into the center of the well resulting in a gradient in the signal across the well. The signal gradient across the well results in signal deterioration and, hence, causes error in determining the relative activity in wells across the plate.
Although, as discussed above, fluorescence measurements benefit from plate materials having minimum intrinsic fluorescence, different screening technique benefit from the optimization of other properties of the microtiter plate material. Such optimization is important when using small microwells. In spectrophotometric techniques, light of a given wave length is directed onto the sample and the amount of light that passes through the sample is detected. Consequently, in this application, it is desirable for the microtiter plate wells to be as transparent as possible so as to minimize the interference with the transmitted light. Luminescence measurements are also used to perform biological assays. In this technique, the light generated by the sample is detected. Since the amount of light generated is relatively small, it is desirable that the microtiter plate material provide as high a reflectance as possible so as to maximize the signal.
Consequently, it would be desirable to provide an apparatus for holding liquids that allowed increased throughput screening by incorporating a large number of small wells but in which liquid was prevented from collecting at the boundaries between adjacent wells. It would also be desirable to provide an apparatus for holding liquids having optimal properties for the particular screening application.